- Email: [email protected]
My Life in Clinical Neuroscience
CHAPTER ONE
My Life in Clinical Neuroscience: The Beginning J.T. Coyle1 McLean Hospital and Harvard Medical School, Belmont, MA, United States 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. The Early Years 3. Medical School 4. Postgraduate Training 5. Getting Started at Hopkins 6. Conclusion References
1 2 3 6 8 10 11
Abstract This chapter recounts the author's life from childhood until he opened his research laboratory as an Assistant Professor in the Department of Pharmacology and Experimental Therapeutics at Johns Hopkins School of Medicine in 1976. It emphasizes the importance of chance opportunities and generous mentoring in the initiation of his career in neuroscience and psychiatric research.
1. INTRODUCTION I am deeply touched and gratified by the generosity of Robbie Schwarcz and Sam Enna in organizing this volume of scientific articles written by former members of my laboratory. And, I thank each of the contributors for taking time from their busy lives to write these wonderful articles. The diversity of topics and the depth of the science are impressive. While we academics focus on the citations to our own publications as a measure of our impact, I think that this view is too restrictive and ignores two important aspects of the scientific endeavor. First, excellent trainees bring to the laboratory different backgrounds, perspectives, and interests that inform and enrich the interactions between mentor and mentee and the resulting Advances in Pharmacology, Volume 76 ISSN 1054-3589 http://dx.doi.org/10.1016/bs.apha.2016.02.002
#
2016 Elsevier Inc. All rights reserved.
1
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research. Second, long after articles by the mentor cease to attract citations, the progeny of the laboratory carry on the scientific mission as so nicely exemplified by these articles (Kanegel, 1993). The invitation to participate in this volume prompted me to reflect on how my career in psychiatry and neuroscience came to be. Instead of a linear trajectory, my career is best characterized by chance events with unclear significance at the time, serendipity, and the wonderful generosity of mentors and colleagues.
2. THE EARLY YEARS I came from a family of physicians. My mother’s father was a smalltown doctor in Iowa. My father was an orthopedic surgeon. Two uncles and two cousins also became physicians. So, I was probably destined to go into medicine. But, I never gave it much thought as I was growing up on the south side of Chicago. In retrospect, I was probably a bit odd as a child. I was not particularly interested in playing sports, did not collect baseball cards, or follow sports teams in spite of the fact that my father was the team physician for the Chicago White Sox, a major league baseball team. I was more interested in how things worked: taking a clock apart at age 6, playing with my Gilbert Chemistry set at 10, raising a polyphemus moth from a caterpillar and struggling with my assignments for J.W. Ellwood’s correspondence course in taxidermy. Adolescence found me in a Jesuit high school hewing to their centuries-honed approach to education—ratio studiorum—that extended over the next 8 years through college at Holy Cross in Worcester, Massachusetts. This curriculum entailed 5 years of classical Greek, 6 years of Latin, and 8 years of French. In addition, my college years were laden with philosophy and theology courses, so that technically I was a French and Philosophy major. The science courses required for medical school were painfully dull, mainly concerned with the memorization of mind-numbing facts or conducting canned experiments. The transformative college experience for me was the junior year that I spent as a student at the Sorbonne, going from a very parochial life at Holy Cross to a very cosmopolitan one in Paris. The return for my last year of college in Worcester was quite painful. At my interview with the admissions committee at Johns Hopkins Medical School, I was asked if I had any experience with research. To some
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puzzled looks, I confidently responded in the affirmative that I did my senior thesis on Samuel Beckett, the Irish play-write, who wrote exclusively in French. But, stepping back now, in spite of this naivety about what was really meant by “research,” I am convinced that this 8-year immersion in literature, languages, and philosophy was extraordinarily helpful in developing the ability to think critically and to communicate effectively during my scientific career. Of course, the experience also greatly altered my view of the world and solidified my interests in the arts, music, and literature. During the summer between college and medical school, I took a job as a psychiatric orderly at the local community hospital since I was vaguely interested in psychiatry as a result of my readings of Freud, Lacan, and Sartre. After a few weeks into the position, the older brother of my closest childhood friend was admitted to the ward with his first episode of schizophrenic psychosis. Soon, I became enmeshed in his paranoid delusions. I then saw psychosis as the ultimate epistemological conundrum but painfully not as abstract as Bishop Berkeley’s hypothesis of immaterialism. This experience cemented my decision to focus on psychiatry in medical school as it seemed to be the best blend of epistemology, humanism, and medicine.
3. MEDICAL SCHOOL Having only a rudimentary background in science, I struggled during the first 2 years of medical school, barely managing a C+ grade point. Nevertheless, the introductory course to Psychiatry in the first year was an extraordinary experience, in spite of the fact that it took place on Saturday mornings. Lecturers included Leon Eisenberg, Jerome Frank, Robert Cooke, Horsley Gantt (a student of Pavlov), and Curt Richter, who discovered circadian rhythms. Contrary to my expectation, the course spent little time on psychoanalytic theory, presenting it as one psychological intervention among many, but rather emphasized empirical and evidence-based approaches to psychiatry. The second year opened with the Pharmacology course. Several lectures were allocated to a new faculty member, Solomon Snyder, who was also a resident in Psychiatry, to cover the nascent subspecialty of psychopharmacology. The topics included stimulants, antidepressants, antipsychotics, and hallucinogens. He reviewed the research on how these drugs exert their effects through altering chemical neurotransmission in the brain. The
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obvious implication of this research was that the etiology of serious mental disorders did not derive from psychic perturbations but rather from biochemical abnormalities. I sought out Dr. Snyder to ask if I could spend the elective quarter in third year in his laboratory. He was pleased to have me, not the least because the extra hands would be quite helpful since he was preoccupied with his psychiatric residency training. The focus of research in the laboratory was characterizing the pharmacology of neurotransmitter transporters in the brain. Julius Axelrod, with whom Snyder had just completed a postdoctoral fellowship at the National Institute of Mental Health, had recently demonstrated that antidepressant drugs act by inhibiting the neuronal reuptake of norepinephrine, thereby potentiating its action at the synapse, a finding that resulted in the Nobel Prize in 1970 (Axelrod, 1971). The primary assay for neurotransmitter uptake used finely chopped rat brain tissue suspended in buffer, which would be allocated to small beakers containing the radioactive neurotransmitter and drugs dissolved in artificial cerebrospinal fluid (Snyder, Green, Hendley, & Gfeller, 1968). My initial task in the laboratory was to screen psychotropic medications for their ability to inhibit the uptake of [3H]norepinephrine. I soon realized that substantial variability was introduced in the assay by the fact that the chopped tissue tended to settle in the pipette, resulting in the early beakers getting more tissue than the later beakers. In a biochemistry laboratory assignment in the first year to study metabolism in dissociated fat cells, I stumbled on some articles discussing how brain tissue could be homogenized in sucrose; and with differential centrifugation, metabolically active pinched-off nerve endings or synaptosomes could be isolated (Marchbanks, 1967; Whittaker, Michaelson, & Kirkland, 1964). I asked Sol if I could try this synaptosome preparation for the transport studies. The preparation had a uniform “milky” appearance, and intra-assay variability declined dramatically, leading to my first peer-reviewed publication (Snyder & Coyle, 1969). That this seemingly minor technical advance came from my insight was very gratifying. Science was no longer boring; it was fun and exciting. Thus, this 10-week experience in Sol’s laboratory dramatically altered the trajectory of my life because he allowed me the freedom to experience the joy of discovery. Over the last 2 years of medical school, I spent 12 months in Sol’s laboratory, part of which was funded by the Denison research scholarship, a real boon as my father had recently passed away. I successfully petitioned the Dean to allow me to work in the laboratory instead of taking a second surgical rotation in the fourth year. Sol was taking a “sabbatical” in the
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Maudsley laboratory of Professor Henry McIlwain, the inventor of the eponymous tissue chopper that was replaced in the Snyder laboratory with synaptosomes. So, supervision was conducted by mail and phone calls in this pre-mail era. In studying the characteristics of catecholamine transport in synaptosomes in various brain regions, I noticed some anomalies between the striatum, the brain region that receives a very dense dopaminergic innervation, and the rest of the brain, which receives noradrenergic innervation (Coyle & Snyder, 1969a). Dopamine transport did not exhibit stereoselectivity in striatal synaptosomes in contrast to [3H] norepinephrine uptake in the cortex. At Sol’s urging, I screened a large number of drugs that were used to treat Parkinson’s disease, many of which were muscarinic receptor antagonists. Benztropine among others clearly differentiated [3H]dopamine transport by striatal synaptosomes from transport for [3H]norepinephrine in other brain regions receiving noradrenergic but not dopaminergic innervation. Published in Science, this was the first description of the dopamine transporter, DAT (Coyle & Snyder, 1969b). In the second year of medical school, a fellow student from my Paris days, who lived in Washington, DC, invited me to a party at her house. Attending the party was this beautiful, smart, and poised woman, Genevieve Sansoucy, who was working on a Masters Degree in Clinical Social Work at Catholic University. I could not forget her. Several months later, my friend invited me to her own wedding; and, thankfully, Genevieve Sansoucy was in attendance. I took her to Baltimore for Chesapeake Bay hard shell crabs for our first date. We were married in the summer of 1968 and took up residence in a fifth floor apartment on Mount Vernon Place in Baltimore. Within 8 years, we went from a couple to a family with three fine sons: Peter, Andrew, and David. Having enjoyed my rotation in pediatrics but also discouraged by my experience with internal medicine rotations, which was dominated by patients primarily suffering from the consequences of poor life choices (drugs, alcohol, tobacco), I decided to pursue postgraduate training in Pediatrics before doing a residency in Psychiatry. I also applied for a postdoctoral position in the Public Health Service at the National Institutes of Health. This position would satisfy my military service requirements, and thus I could avoid joining the armed services at the height of the Vietnam War. I interviewed with Julius Axelrod, Floyd Bloom, Erminio Costa, and the Nobel Prize Laureate Marshal Nirenberg. I accepted Julius Axelrod’s offer for a position in his laboratory, commencing after my pediatric internship at Johns Hopkins Hospital.
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4. POSTGRADUATE TRAINING Starting in Julie’s laboratory after my internship was an unusual experience because he took his vacation during July. This meant that the new postdoctoral fellows had to tag along with established fellows to learn what they were doing and thus get a real sense of the methods and projects of the laboratory. I helped Perry Molinoff (currently Professor of Pharmacology at the University of Pennsylvania) in his project to purify to homogeneity from beef adrenals dopamine-β-hydroxylase (DBH), the final enzyme in the synthesis pathway for norepinephrine. This was my introduction to protein purification and characterization, which was a strength of the Axelrod laboratory. While this project ultimately failed with Perry’s departure, I successfully revisited it when I started my own laboratory at Hopkins (Grzanna & Coyle, 1976; Grzanna, Molliver, & Coyle, 1978). Because of my interest in development, I decided with Julie’s approval to study the development of the noradrenergic system in the rat brain as there was virtually no published information on the maturation of transmitterspecific neuronal systems in the brain, especially prenatally. The project was consistent with catecholaminergic orientation of the laboratory, took advantage of Julie’s skills in enzymology but brought a new theme to the laboratory. A major challenge was to increase the sensitivity of existing assays by 10- to a 100-fold to reliably measure noradrenergic markers as early as 14 days gestation in the rat brain. We were able to show that the synaptosomal uptake of [3H]norepinephrine, the activity of tyrosine hydroxylase, the rate-limiting step in the synthesis of norepinephrine, and the activity DBH all appeared at 15 days gestation, a particularly primitive stage of brain development when the cerebral cortex is in the earliest stages of formation (Coyle & Axelrod, 1971, 1972a, 1972b). Collaborating with David Henry, a fellow in Irwin Kopin’s laboratory, we established the most sensitive assay at the time for measuring norepinephrine by exploiting catechol-O-methyl transferase, an enzyme discovered by Julie, to transfer a [3H]methyl moiety from S-adenosyl methionine ([3H] SAM) to norepinephrine; the product was differentially separated from the [3H]SAM with organic solvents, another of Julie’s strategies (Coyle & Henry, 1973; Fig. 1). As with the other presynaptic markers, norepinephrine appeared in the rat brain at 15 days gestation, suggesting that it might play a role in modulating forebrain development (Coyle, 1977). Based on the simultaneous appearance of all four presynaptic markers, we predicted that
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Fig. 1 Julius Axelrod observing my making a ligation of the rat sciatic nerve to characterize the axoplasmic transport of markers for synaptic vesicles in sympathetic neurons (Wooten & Coyle, 1973).
the locus coeruleus, the primary noradrenergic nucleus in the brain, was formed at 15 days gestation, consistent with the results of [3H]thymidine autoradiography (Lauder & Bloom, 1974). Julie was an extraordinary mentor (Coyle, 2005). Julie’s governmentissued steel desk was strategically placed in the laboratory where it was 4 feet from the reagent scale and 10 feet from the scintillation counter so that every fellow would have to chat with him when either starting or finishing an experiment. Julie made sure his fellows were visible in the field. Every fellow presented a slide talk at the annual American Society for Pharmacology and Experimental Therapeutics meeting, which was the meeting of the year. Julie would turn down seminar invitations and recommend a fellow to speak in his stead. Julie would give fellows journal articles to review. After he felt confident in the quality of these reviews, he would let the fellow sign the review so that soon the journal was soliciting reviews directly from the fellow. Once, I wrote a sarcastic review of what was a weak scientific manuscript. Julie caught me at the reagent scale and said “Joe, a scientific article is like the person’s child. You shouldn’t attack it. Be constructive with your criticism.” I learned humility from a Nobel Prize winner.
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Julie taught us that the best science is not simply confirming your hypotheses but watching out for the anomalous results that may point to novel insights and new, productive directions of inquiry. He suggested that the way to succeed in a research career was to identify an important problem on which few were working. “Life is too short to study uninteresting problems.” He encouraged us to be aggressive in our research—“Be the firstest with the mostest.” He bridled a bit at the introduction of statistics into data analysis, commenting “If you have to do a t-test to prove something is different, it probably isn’t important.” This skepticism about statistics resulted from his uncanny ability to pose experimental questions with such clarity that the results were unequivocal.
5. GETTING STARTED AT HOPKINS During the last year at NIMH, I needed to find a residency in Psychiatry. Although several residency programs expressed interest, Johns Hopkins offered the best opportunity for jump-starting a research laboratory (Fig. 2). Paul Talalay, the Chairman of Pharmacology, working with Joel Elkes, the Chairman of Psychiatry (clearly facilitated by Sol Snyder), offered to appoint
Fig. 2 Solomon Snyder (on right) and me (on left) at the Society for Neuroscience the Julius Axelrod Prize Lecture that I gave at the National Institute of Health (Apr. 15, 2015).
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me as an Assistant Professor of Pharmacology when I started the second year of psychiatric residency and to provide start-up funds and laboratory space. The first person, whom I hired for the laboratory in 1975, was Rob Zaczek as a technician. After serving several years as the senior technician in the laboratory, he completed a PhD in Pharmacology with me (Zaczek et al., 1987) and went on to become the Head of Neuroscience Discovery at BristolMeyer Squibb. Sol kindly referred to me a postdoctoral fellow, who had applied to his laboratory: Robert Schwarcz, PhD. Robbie did not have fellowship support and sold his father’s stamp collection to cover his salary initially. My first RO1 ($26,000 direct costs) concerned using the cultured chick retina to understand dopaminergic neuronal differentiation as model for schizophrenia. Robbie took on this project. He demonstrated the presence of a dopamine sensitive adenylyl cylase along with dopamine and tyrosine hydroxylase but not dopamine-β-hydroxylase in the retina, confirming presence of the retinal dopaminergic neurons (Schwarcz & Coyle, 1976). The retinal project was soon eclipsed by the discovery of in situ excitotoxicity, which arose out of the confluence of two events. Bird and Iversen (1974) had demonstrated in a postmortem neurochemical study of Huntington’s disease (HD) the selective degeneration of striatal intrinsic GABAergic neurons with sparing of the dopaminergic afferents and axons passing through the striatum. In early 1976, Frode Fonnum gave a seminar to the Hopkins Pharmacology Department on the neurotoxic effects of systemic treatment with glutamate in the neonatal rat retina (Karlsen & Fonnum, 1976), presumably mediated by the excitatory effects of the glutamate that accumulated in the eye (Olney, 1969). That seminar prompted my epiphany that direct injection of a glutamate receptor agonist into the rat striatum might replicate the pathology of HD. Mike Kuhar (currently the Candler Professor of Neuropharmacology at Emory University School of Medicine), another one of Sol-trained junior faculty members in the department, was developing a ligand binding assay for excitatory glutamate receptors using the very potent agonist, [3H]kainic acid as the ligand. Mike was able to provide us with kainic acid for our studies. Two micrograms of kainic acid injected into the striatum caused striking rotation away from the side of the injection for 24 h, consistent with over-excitation of the injected striatum. Measurement of presynaptic markers for the striatal GABAergic neurons, glutamic acid decarboxylase (GAD), and cholinergic neurons, choline acetyl transferase (ChaT), revealed
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marked reductions at 48 h after injection, whereas the marker for the dopaminergic terminals, tyrosine hydroxylase, was actually increased by 100%. These preliminary findings supported our hypothesis of neuronal cell body selective and axon sparing effects of in situ injection of a potent glutamate receptor agonist. In short order, Robbie and I prepared detailed letter to Nature describing the dose response, time course, cellular specificity, and histology of the striatal kainate lesion (Coyle & Schwarcz, 1976). The perikaryal-specific excitotoxin lesion to determine the characteristics of neurons whose cell bodies are located at the site of injection became a widely used method cited in over a thousand articles (Coyle & Schwarcz, 1983).
6. CONCLUSION I have focused on these early stages of my career because these experiences and lucky choices set the trajectory for the rest of my life working at the interface between psychiatry and neuroscience. I have tried to ask fundamental questions about how the brain works but always with an eye toward their relevance to neuropsychiatric disorders. One theme that has characterized this search is a focus on the mechanisms responsible for selective neuronal vulnerability in neuropsychiatric disorders including HD (Coyle & Schwarcz, 1976), Alzheimer’s disease (Coyle, Price, & DeLong, 1983), fetal brain damage (Johnston, Grzanna, & Coyle, 1979), Down syndrome (Corsi & Coyle, 1991), amyotrophic lateral sclerosis (Rothstein et al., 1990), and schizophrenia (Balu et al., 2013; Tsai et al., 1995). Another theme has been the role of the excitatory neurotransmitter, glutamic acid, in the pathophysiology of neuropsychiatric disorders that the laboratory has pursued for the last 40 years with over 200 publications on the issue (Coyle, 2006; Coyle, Basu, Benneyworth, Balu, & Konopaske, 2012; Coyle & Puttfarcken, 1993; Herndon & Coyle, 1977; Rothstein et al., 1990; Tsai et al., 1995). What I now cherish are the friendships that evolved from the relationships developed in the laboratory as time erases the distinction between mentor and mentee. Fourteen students did their PhD thesis research in my laboratory, and over 40 postdoctoral fellows and visiting scientists came through the laboratory. Most of these now hold professorial academic positions in the United States, Europe, Canada, and Japan. Perhaps underrecognized are the nearly 30 technicians employed in the laboratory, every one of which went on to obtain a PhD or become a physician such as Angela Guarda, MD (1988), who is now an Associate Professor of Psychiatry at
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Hopkins or Paul Slesinger, PhD (1987–1989), who is now a Professor of Neuroscience at Mount Sinai School of Medicine. I take considerable pride in the accomplishments of all who have come through the laboratory over the last 40 years.
REFERENCES Axelrod, J. (1971). Noradrenaline: Fate and control of its biosynthesis. Science, 173(3997), 598–606. Balu, D. T., Li, Y., Puhl, M. D., Benneyworth, M. A., Basu, A. C., Takagi, S., et al. (2013). Multiple risk pathways for schizophrenia converge in serine racemase knockout mice, a mouse model of NMDA receptor hypofunction. Proceedings of the National Academy of Sciences of the United States of America, 110(26), E2400–E2409. Bird, E. D., & Iversen, L. L. (1974). Huntington’s chorea. Post-mortem measurement of glutamic acid decarboxylase, choline acetyltransferase and dopamine in basal ganglia. Brain, 97(3), 457–472. Corsi, P., & Coyle, J. T. (1991). Nerve growth factor corrects developmental impairments of basal forebrain cholinergic neurons in the trisomy 16 mouse. Proceedings of the National Academy of Sciences of the United States of America, 88(5), 1793–1797. Coyle, J. T. (1977). Biochemical aspects of neurotransmission in the developing brain. International Review of Neurobiology, 20, 65–103. Coyle, J. T. (2005). Julius Axelrod (1912–2004). Molecular Psychiatry, 10(3), 225–226. Coyle, J. T. (2006). Substance use disorders and Schizophrenia: A question of shared glutamatergic mechanisms. Neurotoxicity Research, 10(3–4), 221–233. Coyle, J. T., & Axelrod, J. (1971). Development of the uptake and storage of L-(3H)norepinephrine in the rat brain. Journal of Neurochemistry, 18(11), 2061–2075. Coyle, J. T., & Axelrod, J. (1972a). Dopamine–hydroxylase in the rat brain: Developmental characteristics. Journal of Neurochemistry, 19(2), 449–459. Coyle, J. T., & Axelrod, J. (1972b). Tyrosine hydroxylase in rat brain: Developmental characteristics. Journal of Neurochemistry, 19(4), 1117–1123. Coyle, J. T., Basu, A., Benneyworth, M., Balu, D., & Konopaske, G. (2012). Glutamatergic synaptic dysregulation in schizophrenia: Therapeutic implications. Handbook of Experimental Pharmacology, 213, 267–295. Coyle, J. T., & Henry, D. (1973). Catecholamines in fetal and newborn rat brain. Journal of Neurochemistry, 21(1), 61–67. Coyle, J. T., Price, D. L., & DeLong, M. R. (1983). Alzheimer’s disease: A disorder of cortical cholinergic innervation. Science, 219(4589), 1184–1190. Coyle, J. T., & Puttfarcken, P. (1993). Oxidative stress, glutamate, and neurodegenerative disorders. Science, 262(5134), 689–695. Coyle, J. T., & Schwarcz, R. (1976). Lesion of striatal neurones with kainic acid provides a model for Huntington’s chorea. Nature, 263(5574), 244–246. Coyle, J. T., & Schwarcz, R. (1983). The use of excitatory amino acids as selective neurotoxins. In A. Bjorklund & T. Hokfelt (Eds.), Handbook of chemical neuroanatomy, Vol. 1: Methods in chemical neuroanatomy (pp. 508–527). North-Holland: Elsevier. Coyle, J. T., & Snyder, S. H. (1969a). Catecholamine uptake by synaptosomes in homogenates of rat brain: Stereospecificity in different areas. The Journal of Pharmacology and Experimental Therapeutics, 170(2), 221–231. Coyle, J. T., & Snyder, S. H. (1969b). Antiparkinsonian drugs: Inhibition of dopamine uptake in the corpus striatum as a possible mechanism of action. Science, 166(3907), 899–901.
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Grzanna, R., & Coyle, J. T. (1976). Rat adrenal dopamine-beta-hydroxylase: Purification and immunologic characteristics. Journal of Neurochemistry, 27(5), 1091–1096. Grzanna, R., Molliver, M. E., & Coyle, J. T. (1978). Visualization of central noradrenergic neurons in thick sections by the unlabeled antibody method: A transmitter-specific Golgi image. Proceedings of the National Academy of Sciences of the United States of America, 75, 2502–2506. Herndon, R. M., & Coyle, J. T. (1977). Selective destruction of neurons by a transmitter agonist. Science, 198(4312), 71–72. Johnston, M. V., Grzanna, R., & Coyle, J. T. (1979). Methylazoxymethanol treatment of fetal rats results in abnormally dense noradrenergic innervation of neocortex. Science, 203(4378), 369–371. Kanegel, R. (1993). Apprentice to genius: The making of a scientific dynasty. Baltimore: Johns Hopkins University Press. Karlsen, R. L., & Fonnum, F. (1976). The toxic effect of sodium glutamate on rat retina: Changes in putative transmitters and their corresponding enzymes. Journal of Neurochemistry, 27(6), 1437–1441. Lauder, J. M., & Bloom, F. E. (1974). Ontogeny of monoamine neurons in the locus coeruleus, Raphe nuclei and substantia nigra of the rat. I. Cell differentiation. The Journal of Comparative Neurology, 155(4), 469–481. Marchbanks, R. M. (1967). Compartmentation of acetylcholine in synaptosomes. Biochemical Pharmacology, 16(5), 921–923. Olney, J. W. (1969). Brain lesions, obesity, and other disturbances in mice treated with monosodium glutamate. Science, 164(3880), 719–721. Rothstein, J. D., Tsai, G., Kuncl, R. W., Clawson, L., Cornblath, D. R., Drachman, D. B., et al. (1990). Abnormal excitatory amino acid metabolism in amyotrophic lateral sclerosis. Annals of Neurology, 28(1), 18–25. Schwarcz, R., & Coyle, J. T. (1976). Adenylate cyclase activity in chick retina. General Pharmacology, 7(5), 349–354. Snyder, S. H., & Coyle, J. T. (1969). Regional differences in H3-norepinephrine and H3-dopamine uptake into rat brain homogenates. The Journal of Pharmacology and Experimental Therapeutics, 165(1), 78–86. Snyder, S. H., Green, A., Hendley, E. D., & Gfeller, E. (1968). Noradrenaline: Kinetics of accumulation into slices from different regions of rat brain. Nature, 218(5137), 174–176. Tsai, G., Passani, L. A., Slusher, B. S., Carter, R., Baer, L., Kleinman, J. E., et al. (1995). Abnormal excitatory neurotransmitter metabolism in schizophrenic brains. Archives of General Psychiatry, 52(10), 829–836. Whittaker, V. P., Michaelson, I. A., & Kirkland, R. J. (1964). The separation of synaptic vesicles from nerve-ending particles (‘synaptosomes’). The Biochemical Journal, 90(2), 293–303. Wooten, G. F., & Coyle, J. T. (1973). Axonal transport of catecholamine synthesizing and metabolizing enzymes. Journal of Neurochemistry, 20(5), 1361–1371. Zaczek, R., Arlis, S., Markl, A., Murphy, T., Drucker, H., & Coyle, J. T. (1987). Characteristics of chloride-dependent incorporation of glutamate into brain membranes argue against a receptor binding site. Neuropharmacology, 26(4), 281–287.
My Life in Clinical Neuroscience: The Beginning J.T. Coyle1 McLean Hospital and Harvard Medical School, Belmont, MA, United States 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. The Early Years 3. Medical School 4. Postgraduate Training 5. Getting Started at Hopkins 6. Conclusion References
1 2 3 6 8 10 11
Abstract This chapter recounts the author's life from childhood until he opened his research laboratory as an Assistant Professor in the Department of Pharmacology and Experimental Therapeutics at Johns Hopkins School of Medicine in 1976. It emphasizes the importance of chance opportunities and generous mentoring in the initiation of his career in neuroscience and psychiatric research.
1. INTRODUCTION I am deeply touched and gratified by the generosity of Robbie Schwarcz and Sam Enna in organizing this volume of scientific articles written by former members of my laboratory. And, I thank each of the contributors for taking time from their busy lives to write these wonderful articles. The diversity of topics and the depth of the science are impressive. While we academics focus on the citations to our own publications as a measure of our impact, I think that this view is too restrictive and ignores two important aspects of the scientific endeavor. First, excellent trainees bring to the laboratory different backgrounds, perspectives, and interests that inform and enrich the interactions between mentor and mentee and the resulting Advances in Pharmacology, Volume 76 ISSN 1054-3589 http://dx.doi.org/10.1016/bs.apha.2016.02.002
#
2016 Elsevier Inc. All rights reserved.
1
2
J.T. Coyle
research. Second, long after articles by the mentor cease to attract citations, the progeny of the laboratory carry on the scientific mission as so nicely exemplified by these articles (Kanegel, 1993). The invitation to participate in this volume prompted me to reflect on how my career in psychiatry and neuroscience came to be. Instead of a linear trajectory, my career is best characterized by chance events with unclear significance at the time, serendipity, and the wonderful generosity of mentors and colleagues.
2. THE EARLY YEARS I came from a family of physicians. My mother’s father was a smalltown doctor in Iowa. My father was an orthopedic surgeon. Two uncles and two cousins also became physicians. So, I was probably destined to go into medicine. But, I never gave it much thought as I was growing up on the south side of Chicago. In retrospect, I was probably a bit odd as a child. I was not particularly interested in playing sports, did not collect baseball cards, or follow sports teams in spite of the fact that my father was the team physician for the Chicago White Sox, a major league baseball team. I was more interested in how things worked: taking a clock apart at age 6, playing with my Gilbert Chemistry set at 10, raising a polyphemus moth from a caterpillar and struggling with my assignments for J.W. Ellwood’s correspondence course in taxidermy. Adolescence found me in a Jesuit high school hewing to their centuries-honed approach to education—ratio studiorum—that extended over the next 8 years through college at Holy Cross in Worcester, Massachusetts. This curriculum entailed 5 years of classical Greek, 6 years of Latin, and 8 years of French. In addition, my college years were laden with philosophy and theology courses, so that technically I was a French and Philosophy major. The science courses required for medical school were painfully dull, mainly concerned with the memorization of mind-numbing facts or conducting canned experiments. The transformative college experience for me was the junior year that I spent as a student at the Sorbonne, going from a very parochial life at Holy Cross to a very cosmopolitan one in Paris. The return for my last year of college in Worcester was quite painful. At my interview with the admissions committee at Johns Hopkins Medical School, I was asked if I had any experience with research. To some
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puzzled looks, I confidently responded in the affirmative that I did my senior thesis on Samuel Beckett, the Irish play-write, who wrote exclusively in French. But, stepping back now, in spite of this naivety about what was really meant by “research,” I am convinced that this 8-year immersion in literature, languages, and philosophy was extraordinarily helpful in developing the ability to think critically and to communicate effectively during my scientific career. Of course, the experience also greatly altered my view of the world and solidified my interests in the arts, music, and literature. During the summer between college and medical school, I took a job as a psychiatric orderly at the local community hospital since I was vaguely interested in psychiatry as a result of my readings of Freud, Lacan, and Sartre. After a few weeks into the position, the older brother of my closest childhood friend was admitted to the ward with his first episode of schizophrenic psychosis. Soon, I became enmeshed in his paranoid delusions. I then saw psychosis as the ultimate epistemological conundrum but painfully not as abstract as Bishop Berkeley’s hypothesis of immaterialism. This experience cemented my decision to focus on psychiatry in medical school as it seemed to be the best blend of epistemology, humanism, and medicine.
3. MEDICAL SCHOOL Having only a rudimentary background in science, I struggled during the first 2 years of medical school, barely managing a C+ grade point. Nevertheless, the introductory course to Psychiatry in the first year was an extraordinary experience, in spite of the fact that it took place on Saturday mornings. Lecturers included Leon Eisenberg, Jerome Frank, Robert Cooke, Horsley Gantt (a student of Pavlov), and Curt Richter, who discovered circadian rhythms. Contrary to my expectation, the course spent little time on psychoanalytic theory, presenting it as one psychological intervention among many, but rather emphasized empirical and evidence-based approaches to psychiatry. The second year opened with the Pharmacology course. Several lectures were allocated to a new faculty member, Solomon Snyder, who was also a resident in Psychiatry, to cover the nascent subspecialty of psychopharmacology. The topics included stimulants, antidepressants, antipsychotics, and hallucinogens. He reviewed the research on how these drugs exert their effects through altering chemical neurotransmission in the brain. The
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obvious implication of this research was that the etiology of serious mental disorders did not derive from psychic perturbations but rather from biochemical abnormalities. I sought out Dr. Snyder to ask if I could spend the elective quarter in third year in his laboratory. He was pleased to have me, not the least because the extra hands would be quite helpful since he was preoccupied with his psychiatric residency training. The focus of research in the laboratory was characterizing the pharmacology of neurotransmitter transporters in the brain. Julius Axelrod, with whom Snyder had just completed a postdoctoral fellowship at the National Institute of Mental Health, had recently demonstrated that antidepressant drugs act by inhibiting the neuronal reuptake of norepinephrine, thereby potentiating its action at the synapse, a finding that resulted in the Nobel Prize in 1970 (Axelrod, 1971). The primary assay for neurotransmitter uptake used finely chopped rat brain tissue suspended in buffer, which would be allocated to small beakers containing the radioactive neurotransmitter and drugs dissolved in artificial cerebrospinal fluid (Snyder, Green, Hendley, & Gfeller, 1968). My initial task in the laboratory was to screen psychotropic medications for their ability to inhibit the uptake of [3H]norepinephrine. I soon realized that substantial variability was introduced in the assay by the fact that the chopped tissue tended to settle in the pipette, resulting in the early beakers getting more tissue than the later beakers. In a biochemistry laboratory assignment in the first year to study metabolism in dissociated fat cells, I stumbled on some articles discussing how brain tissue could be homogenized in sucrose; and with differential centrifugation, metabolically active pinched-off nerve endings or synaptosomes could be isolated (Marchbanks, 1967; Whittaker, Michaelson, & Kirkland, 1964). I asked Sol if I could try this synaptosome preparation for the transport studies. The preparation had a uniform “milky” appearance, and intra-assay variability declined dramatically, leading to my first peer-reviewed publication (Snyder & Coyle, 1969). That this seemingly minor technical advance came from my insight was very gratifying. Science was no longer boring; it was fun and exciting. Thus, this 10-week experience in Sol’s laboratory dramatically altered the trajectory of my life because he allowed me the freedom to experience the joy of discovery. Over the last 2 years of medical school, I spent 12 months in Sol’s laboratory, part of which was funded by the Denison research scholarship, a real boon as my father had recently passed away. I successfully petitioned the Dean to allow me to work in the laboratory instead of taking a second surgical rotation in the fourth year. Sol was taking a “sabbatical” in the
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Maudsley laboratory of Professor Henry McIlwain, the inventor of the eponymous tissue chopper that was replaced in the Snyder laboratory with synaptosomes. So, supervision was conducted by mail and phone calls in this pre-mail era. In studying the characteristics of catecholamine transport in synaptosomes in various brain regions, I noticed some anomalies between the striatum, the brain region that receives a very dense dopaminergic innervation, and the rest of the brain, which receives noradrenergic innervation (Coyle & Snyder, 1969a). Dopamine transport did not exhibit stereoselectivity in striatal synaptosomes in contrast to [3H] norepinephrine uptake in the cortex. At Sol’s urging, I screened a large number of drugs that were used to treat Parkinson’s disease, many of which were muscarinic receptor antagonists. Benztropine among others clearly differentiated [3H]dopamine transport by striatal synaptosomes from transport for [3H]norepinephrine in other brain regions receiving noradrenergic but not dopaminergic innervation. Published in Science, this was the first description of the dopamine transporter, DAT (Coyle & Snyder, 1969b). In the second year of medical school, a fellow student from my Paris days, who lived in Washington, DC, invited me to a party at her house. Attending the party was this beautiful, smart, and poised woman, Genevieve Sansoucy, who was working on a Masters Degree in Clinical Social Work at Catholic University. I could not forget her. Several months later, my friend invited me to her own wedding; and, thankfully, Genevieve Sansoucy was in attendance. I took her to Baltimore for Chesapeake Bay hard shell crabs for our first date. We were married in the summer of 1968 and took up residence in a fifth floor apartment on Mount Vernon Place in Baltimore. Within 8 years, we went from a couple to a family with three fine sons: Peter, Andrew, and David. Having enjoyed my rotation in pediatrics but also discouraged by my experience with internal medicine rotations, which was dominated by patients primarily suffering from the consequences of poor life choices (drugs, alcohol, tobacco), I decided to pursue postgraduate training in Pediatrics before doing a residency in Psychiatry. I also applied for a postdoctoral position in the Public Health Service at the National Institutes of Health. This position would satisfy my military service requirements, and thus I could avoid joining the armed services at the height of the Vietnam War. I interviewed with Julius Axelrod, Floyd Bloom, Erminio Costa, and the Nobel Prize Laureate Marshal Nirenberg. I accepted Julius Axelrod’s offer for a position in his laboratory, commencing after my pediatric internship at Johns Hopkins Hospital.
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4. POSTGRADUATE TRAINING Starting in Julie’s laboratory after my internship was an unusual experience because he took his vacation during July. This meant that the new postdoctoral fellows had to tag along with established fellows to learn what they were doing and thus get a real sense of the methods and projects of the laboratory. I helped Perry Molinoff (currently Professor of Pharmacology at the University of Pennsylvania) in his project to purify to homogeneity from beef adrenals dopamine-β-hydroxylase (DBH), the final enzyme in the synthesis pathway for norepinephrine. This was my introduction to protein purification and characterization, which was a strength of the Axelrod laboratory. While this project ultimately failed with Perry’s departure, I successfully revisited it when I started my own laboratory at Hopkins (Grzanna & Coyle, 1976; Grzanna, Molliver, & Coyle, 1978). Because of my interest in development, I decided with Julie’s approval to study the development of the noradrenergic system in the rat brain as there was virtually no published information on the maturation of transmitterspecific neuronal systems in the brain, especially prenatally. The project was consistent with catecholaminergic orientation of the laboratory, took advantage of Julie’s skills in enzymology but brought a new theme to the laboratory. A major challenge was to increase the sensitivity of existing assays by 10- to a 100-fold to reliably measure noradrenergic markers as early as 14 days gestation in the rat brain. We were able to show that the synaptosomal uptake of [3H]norepinephrine, the activity of tyrosine hydroxylase, the rate-limiting step in the synthesis of norepinephrine, and the activity DBH all appeared at 15 days gestation, a particularly primitive stage of brain development when the cerebral cortex is in the earliest stages of formation (Coyle & Axelrod, 1971, 1972a, 1972b). Collaborating with David Henry, a fellow in Irwin Kopin’s laboratory, we established the most sensitive assay at the time for measuring norepinephrine by exploiting catechol-O-methyl transferase, an enzyme discovered by Julie, to transfer a [3H]methyl moiety from S-adenosyl methionine ([3H] SAM) to norepinephrine; the product was differentially separated from the [3H]SAM with organic solvents, another of Julie’s strategies (Coyle & Henry, 1973; Fig. 1). As with the other presynaptic markers, norepinephrine appeared in the rat brain at 15 days gestation, suggesting that it might play a role in modulating forebrain development (Coyle, 1977). Based on the simultaneous appearance of all four presynaptic markers, we predicted that
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Fig. 1 Julius Axelrod observing my making a ligation of the rat sciatic nerve to characterize the axoplasmic transport of markers for synaptic vesicles in sympathetic neurons (Wooten & Coyle, 1973).
the locus coeruleus, the primary noradrenergic nucleus in the brain, was formed at 15 days gestation, consistent with the results of [3H]thymidine autoradiography (Lauder & Bloom, 1974). Julie was an extraordinary mentor (Coyle, 2005). Julie’s governmentissued steel desk was strategically placed in the laboratory where it was 4 feet from the reagent scale and 10 feet from the scintillation counter so that every fellow would have to chat with him when either starting or finishing an experiment. Julie made sure his fellows were visible in the field. Every fellow presented a slide talk at the annual American Society for Pharmacology and Experimental Therapeutics meeting, which was the meeting of the year. Julie would turn down seminar invitations and recommend a fellow to speak in his stead. Julie would give fellows journal articles to review. After he felt confident in the quality of these reviews, he would let the fellow sign the review so that soon the journal was soliciting reviews directly from the fellow. Once, I wrote a sarcastic review of what was a weak scientific manuscript. Julie caught me at the reagent scale and said “Joe, a scientific article is like the person’s child. You shouldn’t attack it. Be constructive with your criticism.” I learned humility from a Nobel Prize winner.
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Julie taught us that the best science is not simply confirming your hypotheses but watching out for the anomalous results that may point to novel insights and new, productive directions of inquiry. He suggested that the way to succeed in a research career was to identify an important problem on which few were working. “Life is too short to study uninteresting problems.” He encouraged us to be aggressive in our research—“Be the firstest with the mostest.” He bridled a bit at the introduction of statistics into data analysis, commenting “If you have to do a t-test to prove something is different, it probably isn’t important.” This skepticism about statistics resulted from his uncanny ability to pose experimental questions with such clarity that the results were unequivocal.
5. GETTING STARTED AT HOPKINS During the last year at NIMH, I needed to find a residency in Psychiatry. Although several residency programs expressed interest, Johns Hopkins offered the best opportunity for jump-starting a research laboratory (Fig. 2). Paul Talalay, the Chairman of Pharmacology, working with Joel Elkes, the Chairman of Psychiatry (clearly facilitated by Sol Snyder), offered to appoint
Fig. 2 Solomon Snyder (on right) and me (on left) at the Society for Neuroscience the Julius Axelrod Prize Lecture that I gave at the National Institute of Health (Apr. 15, 2015).
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me as an Assistant Professor of Pharmacology when I started the second year of psychiatric residency and to provide start-up funds and laboratory space. The first person, whom I hired for the laboratory in 1975, was Rob Zaczek as a technician. After serving several years as the senior technician in the laboratory, he completed a PhD in Pharmacology with me (Zaczek et al., 1987) and went on to become the Head of Neuroscience Discovery at BristolMeyer Squibb. Sol kindly referred to me a postdoctoral fellow, who had applied to his laboratory: Robert Schwarcz, PhD. Robbie did not have fellowship support and sold his father’s stamp collection to cover his salary initially. My first RO1 ($26,000 direct costs) concerned using the cultured chick retina to understand dopaminergic neuronal differentiation as model for schizophrenia. Robbie took on this project. He demonstrated the presence of a dopamine sensitive adenylyl cylase along with dopamine and tyrosine hydroxylase but not dopamine-β-hydroxylase in the retina, confirming presence of the retinal dopaminergic neurons (Schwarcz & Coyle, 1976). The retinal project was soon eclipsed by the discovery of in situ excitotoxicity, which arose out of the confluence of two events. Bird and Iversen (1974) had demonstrated in a postmortem neurochemical study of Huntington’s disease (HD) the selective degeneration of striatal intrinsic GABAergic neurons with sparing of the dopaminergic afferents and axons passing through the striatum. In early 1976, Frode Fonnum gave a seminar to the Hopkins Pharmacology Department on the neurotoxic effects of systemic treatment with glutamate in the neonatal rat retina (Karlsen & Fonnum, 1976), presumably mediated by the excitatory effects of the glutamate that accumulated in the eye (Olney, 1969). That seminar prompted my epiphany that direct injection of a glutamate receptor agonist into the rat striatum might replicate the pathology of HD. Mike Kuhar (currently the Candler Professor of Neuropharmacology at Emory University School of Medicine), another one of Sol-trained junior faculty members in the department, was developing a ligand binding assay for excitatory glutamate receptors using the very potent agonist, [3H]kainic acid as the ligand. Mike was able to provide us with kainic acid for our studies. Two micrograms of kainic acid injected into the striatum caused striking rotation away from the side of the injection for 24 h, consistent with over-excitation of the injected striatum. Measurement of presynaptic markers for the striatal GABAergic neurons, glutamic acid decarboxylase (GAD), and cholinergic neurons, choline acetyl transferase (ChaT), revealed
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marked reductions at 48 h after injection, whereas the marker for the dopaminergic terminals, tyrosine hydroxylase, was actually increased by 100%. These preliminary findings supported our hypothesis of neuronal cell body selective and axon sparing effects of in situ injection of a potent glutamate receptor agonist. In short order, Robbie and I prepared detailed letter to Nature describing the dose response, time course, cellular specificity, and histology of the striatal kainate lesion (Coyle & Schwarcz, 1976). The perikaryal-specific excitotoxin lesion to determine the characteristics of neurons whose cell bodies are located at the site of injection became a widely used method cited in over a thousand articles (Coyle & Schwarcz, 1983).
6. CONCLUSION I have focused on these early stages of my career because these experiences and lucky choices set the trajectory for the rest of my life working at the interface between psychiatry and neuroscience. I have tried to ask fundamental questions about how the brain works but always with an eye toward their relevance to neuropsychiatric disorders. One theme that has characterized this search is a focus on the mechanisms responsible for selective neuronal vulnerability in neuropsychiatric disorders including HD (Coyle & Schwarcz, 1976), Alzheimer’s disease (Coyle, Price, & DeLong, 1983), fetal brain damage (Johnston, Grzanna, & Coyle, 1979), Down syndrome (Corsi & Coyle, 1991), amyotrophic lateral sclerosis (Rothstein et al., 1990), and schizophrenia (Balu et al., 2013; Tsai et al., 1995). Another theme has been the role of the excitatory neurotransmitter, glutamic acid, in the pathophysiology of neuropsychiatric disorders that the laboratory has pursued for the last 40 years with over 200 publications on the issue (Coyle, 2006; Coyle, Basu, Benneyworth, Balu, & Konopaske, 2012; Coyle & Puttfarcken, 1993; Herndon & Coyle, 1977; Rothstein et al., 1990; Tsai et al., 1995). What I now cherish are the friendships that evolved from the relationships developed in the laboratory as time erases the distinction between mentor and mentee. Fourteen students did their PhD thesis research in my laboratory, and over 40 postdoctoral fellows and visiting scientists came through the laboratory. Most of these now hold professorial academic positions in the United States, Europe, Canada, and Japan. Perhaps underrecognized are the nearly 30 technicians employed in the laboratory, every one of which went on to obtain a PhD or become a physician such as Angela Guarda, MD (1988), who is now an Associate Professor of Psychiatry at
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Hopkins or Paul Slesinger, PhD (1987–1989), who is now a Professor of Neuroscience at Mount Sinai School of Medicine. I take considerable pride in the accomplishments of all who have come through the laboratory over the last 40 years.
REFERENCES Axelrod, J. (1971). Noradrenaline: Fate and control of its biosynthesis. Science, 173(3997), 598–606. Balu, D. T., Li, Y., Puhl, M. D., Benneyworth, M. A., Basu, A. C., Takagi, S., et al. (2013). Multiple risk pathways for schizophrenia converge in serine racemase knockout mice, a mouse model of NMDA receptor hypofunction. Proceedings of the National Academy of Sciences of the United States of America, 110(26), E2400–E2409. Bird, E. D., & Iversen, L. L. (1974). Huntington’s chorea. Post-mortem measurement of glutamic acid decarboxylase, choline acetyltransferase and dopamine in basal ganglia. Brain, 97(3), 457–472. Corsi, P., & Coyle, J. T. (1991). Nerve growth factor corrects developmental impairments of basal forebrain cholinergic neurons in the trisomy 16 mouse. Proceedings of the National Academy of Sciences of the United States of America, 88(5), 1793–1797. Coyle, J. T. (1977). Biochemical aspects of neurotransmission in the developing brain. International Review of Neurobiology, 20, 65–103. Coyle, J. T. (2005). Julius Axelrod (1912–2004). Molecular Psychiatry, 10(3), 225–226. Coyle, J. T. (2006). Substance use disorders and Schizophrenia: A question of shared glutamatergic mechanisms. Neurotoxicity Research, 10(3–4), 221–233. Coyle, J. T., & Axelrod, J. (1971). Development of the uptake and storage of L-(3H)norepinephrine in the rat brain. Journal of Neurochemistry, 18(11), 2061–2075. Coyle, J. T., & Axelrod, J. (1972a). Dopamine–hydroxylase in the rat brain: Developmental characteristics. Journal of Neurochemistry, 19(2), 449–459. Coyle, J. T., & Axelrod, J. (1972b). Tyrosine hydroxylase in rat brain: Developmental characteristics. Journal of Neurochemistry, 19(4), 1117–1123. Coyle, J. T., Basu, A., Benneyworth, M., Balu, D., & Konopaske, G. (2012). Glutamatergic synaptic dysregulation in schizophrenia: Therapeutic implications. Handbook of Experimental Pharmacology, 213, 267–295. Coyle, J. T., & Henry, D. (1973). Catecholamines in fetal and newborn rat brain. Journal of Neurochemistry, 21(1), 61–67. Coyle, J. T., Price, D. L., & DeLong, M. R. (1983). Alzheimer’s disease: A disorder of cortical cholinergic innervation. Science, 219(4589), 1184–1190. Coyle, J. T., & Puttfarcken, P. (1993). Oxidative stress, glutamate, and neurodegenerative disorders. Science, 262(5134), 689–695. Coyle, J. T., & Schwarcz, R. (1976). Lesion of striatal neurones with kainic acid provides a model for Huntington’s chorea. Nature, 263(5574), 244–246. Coyle, J. T., & Schwarcz, R. (1983). The use of excitatory amino acids as selective neurotoxins. In A. Bjorklund & T. Hokfelt (Eds.), Handbook of chemical neuroanatomy, Vol. 1: Methods in chemical neuroanatomy (pp. 508–527). North-Holland: Elsevier. Coyle, J. T., & Snyder, S. H. (1969a). Catecholamine uptake by synaptosomes in homogenates of rat brain: Stereospecificity in different areas. The Journal of Pharmacology and Experimental Therapeutics, 170(2), 221–231. Coyle, J. T., & Snyder, S. H. (1969b). Antiparkinsonian drugs: Inhibition of dopamine uptake in the corpus striatum as a possible mechanism of action. Science, 166(3907), 899–901.
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Grzanna, R., & Coyle, J. T. (1976). Rat adrenal dopamine-beta-hydroxylase: Purification and immunologic characteristics. Journal of Neurochemistry, 27(5), 1091–1096. Grzanna, R., Molliver, M. E., & Coyle, J. T. (1978). Visualization of central noradrenergic neurons in thick sections by the unlabeled antibody method: A transmitter-specific Golgi image. Proceedings of the National Academy of Sciences of the United States of America, 75, 2502–2506. Herndon, R. M., & Coyle, J. T. (1977). Selective destruction of neurons by a transmitter agonist. Science, 198(4312), 71–72. Johnston, M. V., Grzanna, R., & Coyle, J. T. (1979). Methylazoxymethanol treatment of fetal rats results in abnormally dense noradrenergic innervation of neocortex. Science, 203(4378), 369–371. Kanegel, R. (1993). Apprentice to genius: The making of a scientific dynasty. Baltimore: Johns Hopkins University Press. Karlsen, R. L., & Fonnum, F. (1976). The toxic effect of sodium glutamate on rat retina: Changes in putative transmitters and their corresponding enzymes. Journal of Neurochemistry, 27(6), 1437–1441. Lauder, J. M., & Bloom, F. E. (1974). Ontogeny of monoamine neurons in the locus coeruleus, Raphe nuclei and substantia nigra of the rat. I. Cell differentiation. The Journal of Comparative Neurology, 155(4), 469–481. Marchbanks, R. M. (1967). Compartmentation of acetylcholine in synaptosomes. Biochemical Pharmacology, 16(5), 921–923. Olney, J. W. (1969). Brain lesions, obesity, and other disturbances in mice treated with monosodium glutamate. Science, 164(3880), 719–721. Rothstein, J. D., Tsai, G., Kuncl, R. W., Clawson, L., Cornblath, D. R., Drachman, D. B., et al. (1990). Abnormal excitatory amino acid metabolism in amyotrophic lateral sclerosis. Annals of Neurology, 28(1), 18–25. Schwarcz, R., & Coyle, J. T. (1976). Adenylate cyclase activity in chick retina. General Pharmacology, 7(5), 349–354. Snyder, S. H., & Coyle, J. T. (1969). Regional differences in H3-norepinephrine and H3-dopamine uptake into rat brain homogenates. The Journal of Pharmacology and Experimental Therapeutics, 165(1), 78–86. Snyder, S. H., Green, A., Hendley, E. D., & Gfeller, E. (1968). Noradrenaline: Kinetics of accumulation into slices from different regions of rat brain. Nature, 218(5137), 174–176. Tsai, G., Passani, L. A., Slusher, B. S., Carter, R., Baer, L., Kleinman, J. E., et al. (1995). Abnormal excitatory neurotransmitter metabolism in schizophrenic brains. Archives of General Psychiatry, 52(10), 829–836. Whittaker, V. P., Michaelson, I. A., & Kirkland, R. J. (1964). The separation of synaptic vesicles from nerve-ending particles (‘synaptosomes’). The Biochemical Journal, 90(2), 293–303. Wooten, G. F., & Coyle, J. T. (1973). Axonal transport of catecholamine synthesizing and metabolizing enzymes. Journal of Neurochemistry, 20(5), 1361–1371. Zaczek, R., Arlis, S., Markl, A., Murphy, T., Drucker, H., & Coyle, J. T. (1987). Characteristics of chloride-dependent incorporation of glutamate into brain membranes argue against a receptor binding site. Neuropharmacology, 26(4), 281–287.